PROJECT SUMMARY/ABSTRACT The long-term goal of the PI?s research program is to elucidate the molecular mechanisms, structure, and regulation of chromatin folding and chromosome condensation. The rationale for pursuing fundamental investigations into DNA folding within nuclei and chromosomes is that this folding goes awry in a number of human diseases, such as the congenital limb malformations brachydactyly, F-syndrome, and polydactyly, and cancers such as glioma and T-cell acute lymphoblastic leukemia. Fundamental studies, such as those proposed here, are needed to fully define the pathophysiology of these diseases and will serve as a basis for developing specific, targeted therapeutics. In pursuit of this long-term goal, the objective during this project period is to define DNA folding within topologically associating domains (TADs), recently discovered independently folded domains of chromatin. TADs are regions of chromatin more likely to interact with themselves than with their neighbors, and are up to 30-fold more condensed than a fully extended, ?beads-on-a-string? chromatin fiber. Specific aims for this project period are: (1) identify intrinsic properties of the chromatin fiber that affect TAD folding, (2) elucidate the folding pattern of DNA within a TAD, and (3) elucidate how TAD structure changes as cells enter mitosis. These aims will be attained using an innovative, holistic approach that integrates methods from molecular genetics, molecular biology, cell biology, and genomics. The expected outcomes of this combined approach is that completion of the proposed work will reveal how DNA is folded within TADs, and how this folding changes as cells exit interphase and enter metaphase. These outcomes will have a positive impact on the field of chromosome biology by providing a better molecular understanding of chromatin folding and chromosome condensation. Such a molecular understanding is expected to ultimately reveal the impact of chromatin folding on gene regulation and cellular heredity. This fundamental knowledge is necessary for defining the pathophysiology of human disease and developing novel therapeutics.